The human immunodeficiency virus (HIV) is a retrovirus that infects cells of the human immune system, leading to acquired immunodeficiency syndrome (AIDS). In 2007, an estimated 33.2 million people worldwide were living with HIV, about 2.7 million people became newly infected, and 2 million patients lost their lives to AIDS. Currently 25 antiretroviral drugs have been approved by the U.S. Food and Drug Administration (FDA) for treating HIV infection, 22 of which are reverse transcriptase inhibitors (RTIs) and protease inhibitors (PIs). RTIs inhibit the activity of HIV reverse transcriptase, a viral DNA polymerase enzyme that HIV needs to reproduce, while PIs inhibit the activity of HIV protease, an enzyme used by the virus to cleave nascent proteins for the final assembly of new virions. Clinical applications of these drugs in different combinations, known as highly active antiretroviral therapy (HAART), have dramatically reduced the morbidity and mortality of AIDS and have significantly improved life expectancy for HIV-infected patients. However, increasing numbers of HIV/AIDS patients on HAART regimens have failed to respond to the current RTIs and PIs due to the emergence of variant strains of drug-resistant HIV. Thus, there is an urgent need for development of new antiretrovirals which are active in inhibiting HIV fusion and entry and against HIV strains that are resistant to current HAART regimens.
Sexual transmission is the most common route of spread of HIV. Both cell-free and cell-associated HIV present in the genital secretions can be sexually transmitted. The continual spread of the HIV epidemic is testament to the lack of safe sex practices. Thus there is an urgent need to develop topically applicable, safe, potent and affordable anti-HIV agent for prevention of HIV sexual transmission.
The event of viral fusion and entry mediated by viral envelope glycoproteins (Env) gp120 and gp41 is the first and most essential step of HIV type 1 (HIV-1) infection. After gp120 binding to the cellular receptor CD4 and a coreceptor, CXCR4 or CCR5, the fusion peptide at the N-terminus of gp41 is exposed, enabling its insertion into the target cell membrane. A series of conformational changes in gp41 take place which lead the protein into its fusogenic state, bringing the viral and target cell membranes into close proximity and promoting membrane fusion.
The core structure of the gp41 ectodomain consists of two 4-3 hydrophobic heptad repeat (HR) regions defined as N-terminal heptad repeat (NHR or HR1, residues 540-590) and C-terminal heptad repeat (CHR or HR2, residues 624-666). Crystallographic studies have shown that CHR can interact with NHR to form a conformation called the “trimer-of-hairpins” or “six-helix bundle” (6-HB), within which three parallel NHRs form a trimeric coiled-coil core and three CHRs pack antiparallelly into the highly conserved hydrophobic grooves along the surface of the inner coiled coil.
Peptides derived from NHR and CHR regions are named NHR- and CHR-peptides, respectively. Some CHR-peptides are potent HIV fusion inhibitors, acting by binding to the viral gp41 inner NHR-trimer to interfere with 6-HB formation. In the early 1990s, the first highly potent CHR-peptide with anti-HIV activity at nanomolar level, SJ-2176, was discovered. Later, two analog peptides, T20 (DP-178) and C34, which inhibited HIV-1-mediated fusion at low nanomolar level were reported. T20 (Fuzeon®; enfuvirtide) has been approved by the US FDA as the first member of a new class of anti-HIV drugs—HIV fusion inhibitors.
T20 is effective as a salvage therapy for HIV/AIDS patients who have failed to respond to current antiretroviral therapeutics, including RTIs and PIs. However, the clinical use of T20 is limited because of its low potency, short half-life, high cost of production and ease with which it induces drug resistance. Using molecular cloning techniques to express recombinant proteins comprising the CHR-sequence fused to a trimerization motif is expected to produce a CHR-trimer with improved potency, relatively long half-life, lower cost of production, and improved resistance profile.
Besides CHR, NHR is also a key target of engineered peptide or protein inhibitors. An NHR-targeted molecule, designed 5-Helix, consists of three NHR helices and two CHR helices interconnected by linkers of five amino acids each. This peptide folds into a structure similar to 6-HB, in which a hydrophobic groove between two NHR-helices is exposed for the binding of viral CHR. 5-Helix shows high potency against HIV-1 infection with a low nanomolar IC50 value. However, it is a difficult task to refold the polypeptide into 5-helix bundle.
In principle, NHR-peptides should also present antiviral activity by targeting the CHR to block the binding of viral CHR to NHR. But the potency of linear NHR-peptides is very low. The reason is that NHR-peptides tend to aggregate in the absence of CHR-peptides. It is supposed that if properly designed NHR-peptides could form stable trimeric coiled-coil conformations that do not aggregate, their efficiency should be as high as that of CHR-peptides.
There are several ways to construct soluble NHR-trimers. Firstly, intermolecular disulfide bonds can be introduced in the NHR-peptide region to stabilize the trimeric conformation. Examples of this method include N34CCG and N35CCG-N13. Secondly, NHR-peptides can be fused to the designed trimetic coiled coils such as portions of GCN4-pIQI (IQ) or IZ to form stable helical trimers, e.g., IQN17, IQN23, and IZN17. These soluble NHR-trimers may inhibit HIV infection as effective as the CHR-peptides.
In the present disclosure, an NHR- or CHR-peptide was linked to a peptide corresponding to the sequence of foldon (Fd), a trimerization motif in the C-terminal domain of T4 fibritin, to form a highly stable and soluble NHR-trimer or CHR-trimer, which are expected to be more potent against HIV infection, more soluble and stable than the monomeric NHR- or CHR-peptides, respectively.